The prospect of developing a gene therapy for the detection and treatment of disease remains high for a number of clinical applications, including cancer, immunodeficiency, and metabolic disorders (Ginn et al., 2013; Peer et al., 2007). Viral-based delivery systems comprise the majority of gene carriers used in gene therapy clinical trials to date; safety concerns, however, motivate the need to engineer alternate delivery systems (Ginn et al., 2013; Yin et al., 2014). Non-viral gene delivery methods have been developed to overcome the main limitations associated with viruses, such as the potential for fatal systemic immune responses, insertional mutagenesis, limited DNA vector size, and issues with large-scale production of viruses (Yin et al., 2014; Baum et al., 2004).
Polymeric nanoparticles are the most widely used non-viral carriers, owing to their protecting the DNA from degradation, and improving intracellular delivery and transfection efficiency of the gene of interest (Bertrand et al., 2005; Mura et al., 2005; Bonnet et al., 2008). Polyelectrolyte complexes (PECs) have been used for drug delivery due to their ability to entrap therapeutic agents.
Of all polymers developed for gene therapy applications, linear polyethylenimine (lPEI) is often utilized because it exhibits high gene delivery efficiency both in vitro and in vivo. Furthermore, compared to its branched PEI counterpart, lPEI has a better safety profile (Bonnet et al., 2008; Jere et al., 2001; Patnaik et al., 2001). It has also been tested in several clinical trials (e.g., NCT01274455 and NCT00595088), primarily through tissue-specific administration routes (Buscail et al., 2015). While there has been increasing efforts to improve the physico-chemical properties and biological performance of lPEI as DNA delivery carrier though molecular engineering and nanoparticle optimization, the progress to clinical application has been hindered by the lack of reproducible and scalable methods for assembly of these complex nanomaterials.
Bulk mixing in the form of vortexing or pipetting are widely used in laboratory environments; but due to their poor micromixing environment, they often lead to high degrees of variability within a preparation batch or between batches as a result of uncontrollable aggregates (Mangraviti et al., 2013; Mangraviti et al., 2015; Mastorakos et al., 2009; Mastorakos et al., 2015; Valencia et al., 2012; Yang et al., 2013; Murday et al., 2009). For example, a recent study reported that batch volume of the nanoparticle preparation by conventional bulk methods significantly affected PEI/siRNA nanoparticle size, with larger preparation solution volumes leading to larger and wider range of particle sizes (Lim et al., 2014). Therefore, developing production methods by which DNA nanoparticles properties can be reproducibly tuned without compromising the biological performance is paramount.
Microfluidic devices with different designs have been reported aiming at delivering better control over particle size and its distribution. For instance, lPEI/siRNA nanoparticles prepared by a microfluidic device displayed significantly higher gene knockdown efficiency in vitro compared to those prepared by bulk mixing (Lim et al., 2014). Furthermore, nanoparticle properties did not change when the batch size of microfluidic mixing was varied. Another study used microfluidics-assisted confinement to prepare polycation/DNA nanocomplexes (Ho et al., 2011). Compared to bulk preparation, microfluidic preparation led to decrease in nanoparticle size distribution, yielding highly condensed, compact and stable nanostructures, reduction in cytotoxicity, and an enhancement of in vitro transfection efficiency. Microfluidic systems, however, can have some limitations, such as the need to formulate complex materials for nanoparticle formulation as well as a limited production capacity (<7.2 g per day) due to the small size of the microfluidic channels (Kolishetti et al., 2010; Romanowsky et al., 2012).
Flash nanoprecipitation (FNP) offers a continuous and scalable process that has been used for the production of block copolymer nanoparticles. This process uses rapid micromixing conditions (on the order of 1 msec) to establish homogeneous supersaturation conditions and controlled precipitation of hydrophobic solutes (organic or inorganic) using block copolymer self-assembly (Johnson et al., Aust. J. Chem., 2003; Johnson et al., Phys. Rev. Lett., 2003; Johnson et al., Aiche J., 2003; Shen et al., 2011). Compared to bulk preparation methods, this process allows for the formation of uniform aggregates with tunable size in a continuous flow operation process, which is amenable for scale-up production. This process also offers a higher degree of versatility and control over particle size and distribution, higher drug encapsulation efficiency, and improved colloidal stability (Shen et al., 2011; D'Addio et al., 2013; D'Addio et al., 2011; D'Addio et al., 2012; Gindy et al., 2008; Lewis et al., 2015; Luo et al., 2014; Santos et al., 2014).
However, in contrast to block copolymer nanoparticles, the assembly of polyelectrolyte complexes is driven by a “complexation reaction”, which is far different from the assembly of amphiphilic copolymers in aqueous media by the FNP method. It appears unlikely that such PEC nanoparticles can be predictably assembled with the FNP method. Hence, there remains a desire in the art to find methods for preparing PEC nanoparticles, which result in properties comparable to those for block copolymer nanoparticles using FNP.